Staphylococcus aureus is now the most common invasive bacterial pathogen in children in the US, and antibiotic resistance rates continue to increase. Novel targets and approaches are needed if a safe and effective S. aureus vaccine is to become a reality. Optimal targets of intervention against S. aureus, a highly human-specific pathogen, may be identified most effectively by defining the antigens that contribute to invasive human infections. LukAB, a secreted leukotoxin, is produced by S. aureus in the setting of invasive human disease and is essential for immune evasion by the pathogen in a variety of models. We have purified a series of potently neutralizing human monoclonal antibodies (mAbs) targeting LukAB, which have broad neutralizing capacity, distinct binding sites and mechanisms of activity, and efficacy in a murine model of sepsis. The overall goals of this proposal are twofold: first, to test the hypothesis that key aspects of the human anti-leukocidin adaptive response include both mechanisms of toxin neutralization and the function of non-neutralizing antibodies targeting these toxins; second, to test the hypothesis that a selected combination of mAbs with distinct functions will most potently inhibit toxin-mediated staphylococcal immune evasion. Careful validation of the anti-leukocidin antibody response in rigorous models will allow for the evaluation of human mAbs as candidate agents of intervention against S. aureus. The proposed studies will define the diversity of the naturally occurring antibody repertoire to S. aureus leukocidins following natural human infection, given their importance in virulence and promise as targets of intervention. This will be accomplished by: 1) Characterizing the diversity and functional capacity of naturally occurring human antibodies targeting the staphylococcal leukocidins following invasive pediatric infection; 2) Elucidating the role of non-neutralizing antibodies specific to S. aureus leukocidins, including antibody-dependent complement deposition and phagocytosis; and 3) Determining the critical components of the anti-leukocidin adaptive response against S. aureus in human blood. We will leverage our existing workflow for the purification and characterization of neutralizing mAbs from prospectively enrolled children with invasive and non-invasive S. aureus infections at a major tertiary care children?s hospital. A panel of human mAbs will be created to allow the investigation of diverse mechanisms of neutralization, cross-toxin activity, and other important functions. The proposed aims will define, for the first time, the breadth of mechanisms by which the human host response targets and neutralizes S. aureus leukocidins during invasive human disease. Together these studies address a critical need for novel anti-S. aureus strategies. This work is designed to produce rationally selected candidates for anti-staphylococcal interventions based on our knowledge of important antigens expressed during human disease, obtained from children with S. aureus infections, to be tested for efficacy in humanized animal models in immediately subsequent work.